Development and validation of innovative sequencing tools for the fast and efficient detection of plant virus

Plant viruses are a major cause of crop losses and decreased agricultural productivity worldwide. Rapid and accurate detection of plant viruses is essential for the implementation of effective control measures. Traditional methods of plant virus detection, such as serological and molecular assays, often present very good performance criteria but they are targeted, and they don’t detect new viruses or divergent strains of known viruses. Overall, developing and validating innovative sequencing tools for fast and efficient detection of plant viruses gained a lot of leverage. Indeed, high throughput sequencing (HTS) tests followed by bioinformatic analyses can detect several viruses at once (including novel ones) and then characterise their genomes. This very high inclusivity allows better monitoring of agricultural pest presence than traditional methods. In addition, the sensitivity of HTS viral detection is theoretically higher than molecular and serological tests, meaning that low-level infection can be traced more efficiently. HTS tests have several drawbacks: the price, the high technical requirements and the cross-contamination of sequences between samples nevertheless. The cost of viral detection by sequencing is higher than traditional methods, but the cost gap is reducing over time as HTS is more and more affordable. More technical skills are required for sequencing and analysis of a sample for virus detection, but the laboratory and bioinformatic protocols are becoming simpler and easier to learn and apply. Cross-contamination between samples is a recurrent phenomenon that is challenging the operational activities of laboratories aiming to detect plant pests. The high sensitivity of HTS has a drawback as it means that cross-contamination is an even more pressing issue than with traditional methods. Cross-contamination is probably one of the main issues when using HTS for viral detection. Indeed, if an unexpected genetic material transfer happens between two samples in the laboratory, one virus can be sequenced in the other sample. Since sequencing sensitivity is high, HTS is more prone to detect this cross-contaminating virus. That may lead to a false positive virus detection (as it is really in the bioinformatic data) while it was not present in the plant. The specificities of HTS technologies (high sensitivity, high inclusivity but with the complexity of laboratory and bioinformatics steps) make their validation difficult compared to traditional tests. Therefore, this thesis describes the side-by-side comparison between traditional tests and HTS technologies for virus indexing of Musa germplasm collection. In addition, an alien control (a specific type of external control) has been used for the first time to II monitor cross-contamination in HTS. In addition, a newly described alien-based filter algorithm, called Cont-ID, has been developed and applied to find the most appropriate limit of detection that should be applied for accurate virus detection taking into account the risk of false negatives and false positives. That way, the detection prediction's confidence can be high enough to be considered for its use in plant virus diagnosis. As written above, HTS technologies can also characterise the genome of the detected viruses. Through variant analysis, the different virus variants can be highlighted. A performance testing was conducted to better understand the difficulties and therefore improve the variants' characterisation. This thesis has therefore addressed several drawbacks limiting potentially the use of HTS technologies for plant virus detection and genome characterisation. It has delivered several milestones to contribute to these technologies' wider and more reliable applications for plant virus detection. Overall, it has reinforced its high potential for improving the control and management of plant virus diseases.2. Zero hunge

Monitoring cross-contamination in viral metagenomic data using an alien-control

editorial reviewedAfter a decade of application in research, high throughput sequencing (HTS) technologies are raising a growing interest for virus diagnostics. Many technical challenges are progressively overcome, but the problem of cross-contamination between samples still needs to be addressed, as it can impact both research and diagnostic fields. Indeed, cross-contamination is an exchange of genetic material between the samples that can occur during the laboratory process and may cause false positive results. In the frame of validating HTS as a diagnostic test for indexing banana viruses, we have used an Alien-control (a wheat sample infected by BYDV, a virus that cannot infect banana) as external control processed together with the analyzed samples. This alien control is a new type of control, allowing to monitor cross-contamination. The monitoring of reads from alien control in banana datasets showed that cross-contamination can be highly variable between sequencing batches and that the contamination threshold should be adapted to each sequencing. In addition, the contamination threshold determination should also consider the results obtained for each banana virus in the batch samples. Those considerations allowed us to reach high performance criteria, e.g. inclusivity, analytical sensitivity, repeatability and reproducibility, of HTS compared to traditional virus indexing on Musa (1). Further on, the automation of cross-contamination checks in bioinformatic analysis via Cont-ID (2) proved that metrics analyses could classify the origin (contamination or infection) of a detected virus with high confidence (91%) for different plants and human datasets. This classification helps raise confidence in the detection and reduce the confirmation work needed by highlighting most critical detections to be checked and also underlined the need for expert judgement. Monitoring viral cross-contamination in metagenomic datasets, whatever its origin, using an alien control-based strategy is possible and should be recommended to raise confidence in results obtained during research projects or diagnostics

Hubble Space Telescope and Ground-Based Observations of the Type Iax Supernovae SN 2005hk and SN 2008A

We present Hubble Space Telescope (HST) and ground-based optical and near-infrared observations of SN 2005hk and SN 2008A, typical members of the Type Iax class of supernovae (SNe). Here we focus on late-time observations, where these objects deviate most dramatically from all other SN types. Instead of the dominant nebular emission lines that are observed in other SNe at late phases, spectra of SNe 2005hk and 2008A show lines of Fe II, Ca II, and Fe I more than a year past maximum light, along with narrow [Fe II] and [Ca II] emission. We use spectral features to constrain the temperature and density of the ejecta, and find high densities at late times, with n_e >~ 10^9 cm^-3. Such high densities should yield enhanced cooling of the ejecta, making these objects good candidates to observe the expected "infrared catastrophe," a generic feature of SN Ia models. However, our HST photometry of SN 2008A does not match the predictions of an infrared catastrophe. Moreover, our HST observations rule out a "complete deflagration" that fully disrupts the white dwarf for these peculiar SNe, showing no evidence for unburned material at late times. Deflagration explosion models that leave behind a bound remnant can match some of the observed properties of SNe Iax, but no published model is consistent with all of our observations of SNe 2005hk and 2008A.Comment: 20 pages, 15 figure

Managing the deluge of newly discovered plant viruses and viroids: an optimized scientific and regulatory framework for their characterization and risk analysis

The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties

Multi-national survey on the methods, efficacy, and safety on the post-approval clinical use of pulsed field ablation (MANIFEST-PF).

AIMS Pulsed field ablation (PFA) is a novel atrial fibrillation (AF) ablation modality that has demonstrated preferential tissue ablation, including no oesophageal damage, in first-in-human clinical trials. In the MANIFEST-PF survey, we investigated the 'real world' performance of the only approved PFA catheter, including acute effectiveness and safety-in particular, rare oesophageal effects and other unforeseen PFA-related complications. METHODS AND RESULTS This retrospective survey included all 24 clinical centres using the pentaspline PFA catheter after regulatory approval. Institution-level data were obtained on patient characteristics, procedure parameters, acute efficacy, and adverse events. With an average of 73 patients treated per centre (range 7-291), full cohort included 1758 patients: mean age 61.6 years (range 19-92), female 34%, first-time ablation 94%, paroxysmal/persistent AF 58/35%. Most procedures employed deep sedation without intubation (82.1%), and 15.1% were discharged same day. Pulmonary vein isolation (PVI) was successful in 99.9% (range 98.9-100%). Procedure time was 65 min (38-215). There were no oesophageal complications or phrenic nerve injuries persisting past hospital discharge. Major complications (1.6%) were pericardial tamponade (0.97%) and stroke (0.4%); one stroke resulted in death (0.06%). Minor complications (3.9%) were primarily vascular (3.3%), but also included transient phrenic nerve paresis (0.46%), and TIA (0.11%). Rare complications included coronary artery spasm, haemoptysis, and dry cough persistent for 6 weeks (0.06% each). CONCLUSION In a large cohort of unselected patients, PFA was efficacious for PVI, and expressed a safety profile consistent with preferential tissue ablation. However, the frequency of 'generic' catheter complications (tamponade, stroke) underscores the need for improvement

La chimica dei nanocomposti e la loro applicazione al restauro dei manoscritti

Cellulose-based artifacts are susceptible to fast degradation due to the presence of detrimental components and to the action of environmental pollutants. As a result, the acidity of pristine material increases, promoting the acid-catalysed depolymerisation of cellulose that alters the mechanical properties of paper. In this paper, the use of innovative dispersions of alkaline earth metal hydroxide nanoparticles will be discussed as a method of counteracting the degradation of paper. The application of the most recent formulations of nanoparticles dispersions for the deacidification of artworks will be highlighted. Finally, the usage of innovative gel formulations for the cleaning of cellulose-based artworks will be discussed

Detection of single nucleotide polymorphisms in virus genomes assembled from high-throughput sequencing data: large-scale performance testing of sequence analysis strategies

peer reviewedRecent developments in high-throughput sequencing (HTS) technologies and bioinformatics have drastically changed research in virology, especially for virus discovery. Indeed, proper monitoring of the viral population requires information on the different isolates circulating in the studied area. For this purpose, HTS has greatly facilitated the sequencing of new genomes of detected viruses and their comparison. However, bioinformatics analyses allowing reconstruction of genome sequences and detection of Single Nucleotide Polymorphisms (SNPs) can potentially create bias and has not been widely addressed so far. Therefore, more knowledge is required on the limitations of predicting SNPs based on HTS-generated sequence samples. To address this issue, we compared the ability of 14 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 21 variants of pepino mosaic virus (PepMV) in three samples through large-scale Performance-Testing (PT) using three artificially designed datasets. To evaluate the impact of bioinformatics analyses, they were divided into three key steps: reads pre-processing, virus-isolate identification, and variant calling. Each step was evaluated independently through an original, PT design including discussion and validation between participants at each step. Overall, this work underlines key parameters influencing SNPs detection and proposes recommendations for reliable variant calling for plant viruses. The identification of the closest reference, mapping parameters and manual validation of the detection were recognized as the most impactful analysis steps for the success of the SNPs detections. Strategies to improve the prediction of SNPs are also discussed